Epstein-Barr Virus Nuclear Protein Research Articles

Exploring the Genetic Diversity of Epstein-Barr Virus among Patients with Gastric Cancer in Southern Chile.

The Epstein-Barr virus (EBV) has been associated with gastric cancer (GC), one of the deadliest malignancies in Chile and the world. Little is known about Chilean EBV strains. This study aims to investigate the frequency and genetic diversity of EBV in GC in patients in southern Chile. To evaluate the prevalence of EBV in GC patients from the Chilean population, we studied 54 GC samples using the gold standard detection method of EBV-encoded small RNA (EBER). The EBV-positive samples were subjected to amplification and sequencing of the Epstein-Barr virus nuclear protein 3A (EBNA3A) gene to evaluate the genetic diversity of EBV strains circulating in southern Chile. In total, 22.2% of the GC samples were EBV-positive and significantly associated with diffuse-type histology (p = 0.003). Phylogenetic analyses identified EBV-1 and EBV-2 in the GC samples, showing genetic diversity among Chilean isolates. This work provides important information for an epidemiological follow-up of the different EBV subtypes that may cause GC in southern Chile.

Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over “classical” rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo.

Nucleolar c-Myc recruitment by a Vibrio T3SS effector promotes host cell proliferation and bacterial virulence.

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V.parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.

EBP2, a novel NPM-ALK-interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53.

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large-cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0 /G1 -phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt-mTORC1 pathway in NPM-ALK-positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.

Correction: Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

[This corrects the article DOI: 10.1371/journal.ppat.1006890.].

Epstein-Barr virus nuclear antigen EBNA-LP is essential for transforming naïve B cells, and facilitates recruitment of transcription factors to the viral genome.

The Epstein-Barr virus (EBV) nuclear antigen leader protein (EBNA-LP) is the first viral latency-associated protein produced after EBV infection of resting B cells. Its role in B cell transformation is poorly defined, but it has been reported to enhance gene activation by the EBV protein EBNA2 in vitro. We generated EBNA-LP knockout (LPKO) EBVs containing a STOP codon within each repeat unit of internal repeat 1 (IR1). EBNA-LP-mutant EBVs established lymphoblastoid cell lines (LCLs) from adult B cells at reduced efficiency, but not from umbilical cord B cells, which died approximately two weeks after infection. Adult B cells only established EBNA-LP-null LCLs with a memory (CD27+) phenotype. Quantitative PCR analysis of virus gene expression after infection identified both an altered ratio of the EBNA genes, and a dramatic reduction in transcript levels of both EBNA2-regulated virus genes (LMP1 and LMP2) and the EBNA2-independent EBER genes in the first 2 weeks. By 30 days post infection, LPKO transcription was the same as wild-type EBV. In contrast, EBNA2-regulated cellular genes were induced efficiently by LPKO viruses. Chromatin immunoprecipitation revealed that EBNA2 and the host transcription factors EBF1 and RBPJ were delayed in their recruitment to all viral latency promoters tested, whereas these same factors were recruited efficiently to several host genes, which exhibited increased EBNA2 recruitment. We conclude that EBNA-LP does not simply co-operate with EBNA2 in activating gene transcription, but rather facilitates the recruitment of several transcription factors to the viral genome, to enable transcription of virus latency genes. Additionally, our findings suggest that EBNA-LP is essential for the survival of EBV-infected naïve B cells.

New Interactors of the Truncated EBNA-LP Protein Identified by Mass Spectrometry in P3HR1 Burkitt's Lymphoma Cells.

The Epstein-Barr virus nuclear antigen leader protein (EBNA-LP) acts as a co-activator of EBNA-2, a transcriptional activator essential for Epstein-Barr virus (EBV)-induced B-cell transformation. Burkitt’s lymphoma (BL) cells harboring a mutant EBV strain that lacks both the EBNA-2 gene and 3′ exons of EBNA-LP express Y1Y2-truncated isoforms of EBNA-LP (tEBNA-LP) and better resist apoptosis than if infected with the wild-type virus. In such BL cells, tEBNA-LP interacts with the protein phosphatase 2A (PP2A) catalytic subunit (PP2A C), and this interaction likely plays a role in resistance to apoptosis. Here, 28 cellular and four viral proteins have been identified by mass spectrometry as further possible interactors of tEBNA-LP. Three interactions were confirmed by immunoprecipitation and Western blotting, namely with the A structural subunit of PP2A (PP2A A), the structure-specific recognition protein 1 (SSRP1, a component of the facilitate chromatin transcription (FACT) complex), and a new form of the transcription factor EC (TFEC). Thus, tEBNA-LP appears to be involved not only in cell resistance to apoptosis through its interaction with two PP2A subunits, but also in other processes where its ability to co-activate transcriptional regulators could be important.

Immuno-PCR Assay for Quantitation of Antibodies to Epstein-Barr Virus

Successful disease prevention and therapy critically depend on timely diagnosis of infections. Quantitative immuno-PCR (qiPCR) technology improves the sensitivity in the detection of antibodies to pathogens. A qiPCR-based assay was developed to determine IgG antibodies to Epstein-Barr virus (EBV) in the human blood serum. EBV nuclear protein 1 fragment (pEBV) was expressed in Escherichia coli. A synthetic single-stranded deoxyribonucleotide was conjugated to streptavidin, and the conjugate was used to detect рEBV-IgG1-biotin complexes by qiPCR. The IgG1 titers determined by qiPCR were compared to the results of enzyme-linked immunosorbent assay (ELISA). The sensitivity of qiPCR was one order of magnitude higher than that of ELISA. Thus, a highly sensitive qiPCR-based assay was developed to quantitate antibodies specific to the recombinant EBV antigen.

Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells.

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma.

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A- or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A- and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro. EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity.

The EBNA3 family of Epstein-Barr virus nuclear proteins associates with the USP46/USP12 deubiquitination complexes to regulate lymphoblastoid cell line growth.

The Epstein-Barr virus (EBV) nuclear proteins EBNA3A, EBNA3B, and EBNA3C interact with the cell DNA binding protein RBPJ and regulate cell and viral genes. Repression of the CDKN2A tumor suppressor gene products p16INK4A and p14ARF by EBNA3A and EBNA3C is critical for EBV mediated transformation of resting B lymphocytes into immortalized lymphoblastoid cell lines (LCLs). To define the composition of endogenous EBNA3 protein complexes, we generated lymphoblastoid cell lines (LCLs) expressing flag-HA tagged EBNA3A, EBNA3B, or EBNA3C and used tandem affinity purification to isolate each EBNA3 complex. Our results demonstrated that each EBNA3 protein forms a distinct complex with RBPJ. Mass-spectrometry revealed that the EBNA3A and EBNA3B complexes also contained the deubquitylation complex consisting of WDR48, WDR20, and USP46 (or its paralog USP12) and that EBNA3C complexes contained WDR48. Immunoprecipitation confirmed that EBNA3A, EBNA3B, and EBNA3C association with the USP46 complex. Using chromatin immunoprecipitation, we demonstrate that WDR48 and USP46 are recruited to the p14ARF promoter in an EBNA3C dependent manner. Mapping studies were consistent with WDR48 being the primary mediator of EBNA3 association with the DUB complex. By ChIP assay, WDR48 was recruited to the p14ARF promoter in an EBNA3C dependent manner. Importantly, WDR48 associated with EBNA3A and EBNA3C domains that are critical for LCL growth, suggesting a role for USP46/USP12 in EBV induced growth transformation.

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve

Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.

Repression of the Proapoptotic CellularBIK/NBKGene by Epstein-Barr Virus Antagonizes Transforming Growth Factor β1-Induced B-Cell Apoptosis

The Epstein-Barr virus (EBV) establishes a lifelong latent infection in humans. EBV infection of primary B cells causes cell activation and proliferation, a process driven by the viral latency III gene expression program, which includes EBV nuclear proteins (EBNAs), latent membrane proteins, and untranslated RNAs, including microRNAs. Some latently infected cells enter the long-lived memory B-cell compartment and express only EBNA1 transiently (Lat I) or no EBV protein at all (Lat 0). Targeting the molecular machinery that controls B-cell fate decisions, including the Bcl-2 family of apoptosis-regulating proteins, is crucial to the EBV cycle of infection. Here, we show that BIK (also known as NBK), which encodes a proapoptotic "sensitizer" protein, is repressed by the EBNA2-driven Lat III program but not the Lat I program. BIK repression occurred soon after infection of primary B cells by EBV but not by a recombinant EBV in which the EBNA2 gene had been knocked out. Ectopic BIK induced apoptosis in Lat III cells by a mechanism dependent on its BH3 domain and the activation of caspases. We show that EBNA2 represses BIK in EBV-negative B-cell lymphoma-derived cell lines and that this host-virus interaction can inhibit the proapoptotic effect of transforming growth factor β1 (TGF-β1), a key physiological mediator of B-cell homeostasis. Reduced levels of TGF-β1-associated regulatory SMAD proteins were bound to the BIK promoter in response to EBV Lat III or ectopic EBNA2. These data are evidence of an additional mechanism used by EBV to promote B-cell survival, namely, the transcriptional repression of the BH3-only sensitizer BIK. Over 90% of adult humans are infected with the Epstein-Barr virus (EBV). EBV establishes a lifelong silent infection, with its DNA residing in small numbers of blood B cells that are a reservoir from which low-level virus reactivation and shedding in saliva intermittently occur. Importantly, EBV DNA is found in some B-cell-derived tumors in which viral genes play a key role in tumor cell emergence and progression. Here, we report for the first time that EBV can shut off a B-cell gene called BIK. When activated by a molecular signal called transforming growth factor β1 (TGF-β1), BIK plays an important role in killing unwanted B cells, including those infected by viruses. We describe the key EBV-B-cell molecular interactions that lead to BIK shutoff. These findings further our knowledge of how EBV prevents the death of its host cell during infection. They are also relevant to certain posttransplant lymphomas where unregulated cell growth is caused by EBV genes.

Gene variations of Epstein-Barr virus nuclear antigen 3A in nasopharyngeal carcinomas, gastric carcinomas and healthy carriers in northern China

The Epstein-Barr virus (EBV) nuclear antigen protein 3A (EBNA-3A), a protein of 944 amino acids, is one of five EBNAs (EBNA-1, -2, -LP, -3A and -3C) essential for conversion of primary B lymphocytes to lymphoblastoid cell lines. To characterize the variations of the EBNA-3A gene and explore the association between EBNA-3A gene variations and EBV-associated diseases, we sequenced the key regions of EBNA-3A in the isolates of 30 EBV-associated gastric carcinomas (EBVaGCs), 44 nasopharyngeal carcinomas (NPCs) and 48 samples from healthy donors in northern China. We found that EBNA-3A shares a common evolutionary origin with isolates from southern China and Japan but has the character of a geographical variant. Based on a phylogenetic tree, all of the samples can be subdivided into three patterns, named 3A-8, 3A-5 and B95-8-like. The distribution of EBNA-3A subtypes among EBVaGC, NPC and healthy donors is not significantly different. The subtype 3A-8 is predominant not only in northern China but also in southern China; it is a geographically associated polymorphism in China.

Abortive lytic Epstein–Barr virus replication in tonsil-B lymphocytes in infectious mononucleosis and a subset of the chronic fatigue syndrome

Correspondence: A Martin Lerner 32804 Pierce Road, Beverly Hills, MI, 48025, USA Tel +1 248 540 9866 Fax +1 248 540 0139 Email amartinlerner@yahoo.com Abstract: A systematic 2001–2007 review of 142 chronic fatigue syndrome (CFS) patients identified 106 CFS patients with elevated serum IgG antibodies to the herpesviruses Epstein– Barr virus (EBV), cytomegalovirus, or human herpesvirus (HHV) 6 in single or multiple infections, with no other co-infections detected. We named these 106 patients group-A CFS. Eighty-six of these 106 group-A CFS patients (81%) had elevated EBV early antibody, early antigen (diffuse), serum titers. A small group of six patients in the group-A EBV subset of CFS, additionally, had repetitive elevated-serum titers of antibody to the early lytic replicationencoded proteins, EBV dUTPase, and EBV DNA polymerase. The presence of these serum antibodies to EBV dUTPase and EBV DNA polymerase indicated EBV abortive lytic replication in these 6 CFS patients. None of 20 random control people (ageand sex-matched, with blood drawn at a commercial laboratory) had elevated serum titers of antibody to EBV dUTPase or EBV DNA polymerase (P , 0.01). This finding needs verification in a larger group of EBV CFS subset patients, but if corroborated, it may represent a molecular marker for diagnosing the EBV subset of CFS. We review evidence that EBV abortive lytic replication with unassembled viral proteins in the blood may be the same in infectious mononucleosis (IM) and a subset of CFS. EBV-abortive lytic replication in tonsil plasma cells is dominant in IM. No complete lytic virion is in the blood of IM or CFS patients. Complications of CFS and IM include cardiomyopathy and encephalopathy. Circulating abortive lytic-encoded EBV proteins (eg, EBV dUTPase, EBV DNA polymerase, and others) may be common to IM and CFS. The intensity and duration of the circulating EBV-encoded proteins might differentiate the IM and EBV subsets of CFS. Abortive lytic replication may be a pathogenic mechanism for EBV disease. EBV (HHV4) is a gamma herpesvirus composed of dsDNA about 170 Kb in length. For this discussion, there are early genes (including expressions of encoded proteins EBV dUTPase, DNA polymerase, and nuclear proteins) and late genes (including expressions of capsid and membrane proteins). Abortive infection infers incomplete virion expressions of either early or late proteins, but the virion is incomplete. The lytic virus infers a complete virion. The pathologic consequences of EBV abortive replication are currently being investigated by authors.

Similarities between the Epstein-Barr Virus (EBV) Nuclear Protein EBNA1 and the Pioneer Transcription Factor FoxA: Is EBNA1 a "Bookmarking" Oncoprotein that Alters the Host Cell Epigenotype?

EBNA1, a nuclear protein expressed in all EBV-associated neoplasms is indispensable for the maintenance of the viral episomes in latently infected cells. EBNA1 may induce genetic alterations by upregulating cellular recombinases, production of reactive oxygen species (ROS) and affecting p53 levels and function. All these changes may contribute to tumorigenesis. In this overview we focus, however, on the epigenetic alterations elicited by EBNA1 by drawing a parallel between EBNA1 and the FoxA family of pioneer transcription factors. Both EBNA1 and FoxA induce local DNA demethylation, nucleosome destabilization and bind to mitotic chromosomes. Local DNA demethylation and nucleosome rearrangement mark active promoters and enhancers. In addition, EBNA1 and FoxA, when associated with mitotic chromatin may “bookmark” active genes and ensure their reactivation in postmitotic cells (epigenetic memory). We speculate that DNA looping induced by EBNA1-EBNA1 interactions may reorganize the cellular genome. Such chromatin loops, sustained in mitotic chromatin similarly to the long-distance interactions mediated by the insulator protein CTCF, may also mediate the epigenetic inheritance of gene expression patterns. We suggest that EBNA1 has the potential to induce patho-epigenetic alterations contributing to tumorigenesis.

Effect of Rheumatoid Arthritis (RA) Susceptibility Genes on the Immune Response to Viral Citrullinated Peptides in RA

To the Editor: Anticitrullinated peptide/protein antibodies (ACPA) are a family of rheumatoid arthritis (RA)-specific antibodies that recognize various protein sequences containing citrulline, the deiminated form of arginine produced posttranslationally by peptidylarginine deiminase (PAD) enzymes. Recently, viral citrullinated peptides (VCP) derived from Epstein-Barr virus nuclear proteins EBNA-1 and EBNA-2 have been described as a target of ACPA in RA sera1,2. Genetic factors predisposing to RA, namely HLA-DRB1 shared epitope (SE) alleles and PTPN22 gene variants, as well as the TRAF1/C5 locus and PADI4 susceptible haplotype, have been found to be predominantly associated with ACPA-positive RA, even if the association with ACPA-negative RA cannot be excluded. We evaluated the influence of genes conferring susceptibility to RA on the production of anti-VCP antibodies and tested the interaction of VCP with HLA in vitro . We analyzed 172 patients with RA of French white origin3. All provided informed consent and the Ethics Committee of Hopital Bicetre (AP-HP, Paris, France) approved the study. HLA-DRB1 typing and subtyping were performed by a polymerase chain reaction (PCR)-based method, using a panel of sequence-specific oligonucleotide probes. PADI4 haplotypes were obtained by typing 3 single-nucleotide polymorphisms (SNP: rs1886303, PADI4\_92, PADI4\_96 – CGC haplotype) by PCR-restriction fragment-length polymorphism (RFLP)4. PTPN22 (R620W; rs24766601 – T variant) and C5/TRAF1 region polymorphisms (rs10818488 – A variant) were genotyped by PCR-RFLP5,6. … Address correspondence to Dr. P. Migliorini; E-mail: paola.migliorini{at}med.unipi.it

Characterization of Epstein-Barr virus type 1 nuclear antigen 3C sequence patterns of nasopharyngeal and gastric carcinomas in northern China

Epstein-Barr virus nuclear antigen protein 3C (EBNA3C) is a 992-amino-acid protein that has been shown to play a complex regulatory role in the transcription of viral and cellular genes. In this study, we successfully amplified 26 Epstein-Barr virus (EBV)-associated gastric carcinomas (EBVaGCs), 50 nasopharyngeal carcinomas (NPCs) and 27 throat washing (TW) samples from healthy donors. Based on a phylogenetic tree, the samples could be divided into three patterns. 3C-6 was the predominant subtype in northern China, and the variations between the strains sequenced in our study and those from southern China and Japan were similar, but differences were also identified. The distribution of EBNA3C subtypes among EBVaGCs, NPCs and healthy donors was not significantly different. These data suggest that EBNA3C gene variations are geographically restricted rather than tumor-specific polymorphisms.

Contributions of the Epstein-Barr Virus EBNA1 Protein to Gastric Carcinoma

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.

Epstein–Barr virus nuclear protein 3C binds to the N-terminal (NTD) and beta trefoil domains (BTD) of RBP/CSL; Only the NTD interaction is essential for lymphoblastoid cell growth

Association of EBV nuclear proteins EBNA2, EBNA3A and EBNA3C with RBP/CSL, is essential for lymphoblastoid cell line (LCL) proliferation. Conserved residues in the EBNA3 homology domain, required for RBP/CSL interaction, lack the WΦP motif that mediates EBNA2 and Notch binding to the RBP/CSL beta-trefoil domain (BTD). We map RBP/CSL interacting residues within EBNA3A(aa128–204) and EBNA3C(aa211–233). The EBNA3A results are consistent with an earlier report (aa125–222), but the EBNA3C domain is unexpectedly small and includes a “WTP” sequence. This EBNA3C WTP motif confers RBP/CSL binding in vitro, in yeast, and in mammalian cells. Further, an EBNA3C WTP → STP(W227S) mutation impaired BTD binding whereas EBNA3 homology domain mutations disrupted RBP/CSL N-terminal domain (NTD) binding. WTP was not essential for EBNA3C repression of EBNA2 in reporter assays or for maintenance of LCL growth. Our results indicate that EBNA3 proteins interact with multiple RBP/CSL domains, but only NTD interactions are required for LCL growth.